Polymerase incorporation of pyrene-nucleoside triphosphates

Article abstract of DOI:10.1016/j.bmcl.2012.04.101

Pyrene-deoxynucleoside triphosphates (dPTPs), varying by the positioning of the aromatic system, were synthesized. Their ability to function as substrates for the Klenow fragment of Escherichia coli DNA polymerase I and the TdT polymerase was assessed. The dPTPs are all equally well tolerated by the Klenow fragment, and lead to elongation of up to 5 extra nucleotides of a ssDNA primer in a TdT-mediated reaction. The tailing efficiency of the dPTPs compares favorably to other less drastically modified dNTPs.

Full text of DOI:10.1016/j.bmcl.2012.04.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4428–4430
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Polymerase incorporation of pyrene-nucleoside triphosphates
⇑
Marcel Hollenstein , Filip Wojciechowski, Christian J. Leumann
Department of Chemistry & Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrene-deoxynucleoside triphosphates (dPTPs), varying by the positioning of the aromatic system, were
synthesized. Their ability to function as substrates for the Klenow fragment of Escherichia coli DNA poly-
merase I and the TdT polymerase was assessed. The dPTPs are all equally well tolerated by the Klenow
fragment, and lead to elongation of up to 5 extra nucleotides of a ssDNA primer in a TdT-mediated reac-
tion. The tailing efficiency of the dPTPs compares favorably to other less drastically modified dNTPs.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 1 April 2012
Revised 20 April 2012
Accepted 21 April 2012
Available online 5 May 2012
Keywords:
Nucleosides
DNA
Terminal deoxynucleotidyl
transferase (TdT)
Nucleotides
Triphosphate analogues
Nucleoside triphosphates (dNTPs) have emerged as a versatile
and wide-ranging platform for the generation of chemically modi-
fied oligonucleotides.1–3 Indeed, a myriad of dNTPs has been gener-
ated to supplement the rather limited chemical arsenal of the
nucleobases, especially in the context of in vitro selections for
the development of DNAzymes.4–10 Moreover, modified dNTPs
have been designed for a wide-ranging palette of applications
including electrochemical and fluorescent labeling of nucleic
acids,11–15,1,16 bar-coding of DNA,17 and bioanalytical purposes.18,2
In particular, modified dNTPs act as a pivotal tool for the discovery
of unnatural base pair systems19,20 and by the same token, for
investigating the contribution of electrostatic forces, aromatic
stacking, and H-bonding to the strength of the Watson–Crick base
pairs.21,22 In this context, 1-pyrene deoxynucleoside triphosphate
d1PTP 1 (Fig. 1) was originally conceived to evaluate the impor-
tance of these factors on the fidelity of replication of DNA polymer-
ases.21 The modified triphosphate d1PTP revealed to be a potent
substrate for the Klenow fragment of Escherichia coli DNA polymer-
ase I, thus underscoring the importance of steric complementarity.
Terminal deoxynucleotidyl transferase (TdT) is a Co2+-depen-
dent DNA polymerase that catalyzes the stepwise homopolymer-
ization of nucleoside triphosphates on single-stranded
oligonucleotides.23 Surprisingly, the potential of this polymerase
to incorporate modified dNTPs is vastly unexplored and only a
few scarce examples have been reported so far.24,25
from the original d1PTP 1 by the position of the anchoring point con-
necting the aromatic residue to the sugar unit, thus causing a differ-
ential tilt of the pyrene, which in turn could possibly lead to altered
stacking abilities. We then explored the polymerase uptake of these
modified dNTPs with a special emphasis on the TdT enzyme.
The synthesis of the modified triphosphates 1–3 proceeded
smoothly and is highlighted in Scheme 1. Briefly, the DMT-pro-
tected nucleoside precursors 4–626 were 30-O-acetylated and the
masking trityl groups were removed by treatment with dichloro-
acetic acid (see Supplementary data). The resulting intermediates
10–12 were then converted to the corresponding triphosphates
by application of the one pot-four steps procedure developed by
Ludwig and Eckstein.27 RP-HPLC purification led to isolation of
the pure dNTPs in moderate yields (11–36%).
With the modified triphosphates in hand, we first investigated
their uptake by the Klenow fragment of Escherichia coli DNA
polymerase I (Fig. 2 and Fig. S1, Supplementary data). Indeed, using
a 28-mer template containing a tetrahydrofuran abasic analogue
(/),21 standing start (Fig. S1, Supplementary data) and running
start (Fig. 2) primer extension reactions were carried out.
All the modified dNTPs are good substrates for the Klenow frag-
ment, since both in the running start and the standing start exper-
iments, efficient extensions could be observed, with the dPTPs
placed opposite to the abasic sites. However, the position of the
aromatic system on the deoxyribose has little impact on the uptake
efficacy. Indeed, none of the triphosphates showed any differential
propensity at incorporation and all the running start experiments
stalled after incorporation of the dPMPs.
Here, we report on the synthesis of two novel pyrene-deoxynu-
cleoside triphosphates d2PTP 2 and d4PTP 3 (Fig. 1), which differ
The dPTPs were then tested for their ability to act as substrates
⇑
Corresponding author. Tel.: +41 31 631 4372; fax: +41 31 631 3422.
E-mail address: hollenstein@dcb.unibe.ch (M. Hollenstein).
in TdT-catalyzed extension reactions.28 In order to do so, a 15-mer
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.101

M. Hollenstein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4428–4430
4429
4
3
2
1
4-O9P3O
4-O9P3O
4-O9P3O
O
O
O
OH
OH
OH
1
2
3
Figure 1. Chemical structures of the modified triphosphates d1PTP (1), d2PTP (2), and d4PTP (3). The pyrene numbering system is shown on dPTP 1.
Ar
Ar
HO
Ar
DMTrO
c)
DMTrO
b)
O
a)
O
O
1
,
2 3
,
OAc
OAc
OH
; Ar = 1Py
; Ar = 1Py
; Ar = 1Py
10
11
7
8
4
5
; Ar = 2Py
; Ar = 2Py
; Ar = 2Py
12; Ar = 4Py
9; Ar = 4Py
6; Ar = 4Py
Scheme 1. Reagents and conditions: (a) Ac2O, pyridine, 25 °C, 12 h, 7: 99%, 8: quant., 9: 98%; (b) DCAA (1%), DCM, 25 °C, 40 min, 10: 98%, 11: 88%, 12: 77%; (c) (i) 2-chloro-
1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, rt, 45 min, (ii) (nBu3NH)2H2P2O7, DMF, nBu3N, rt, 40 min, (iii) I2, pyridine, H2O, rt, 30 min, (iv) NH3(aq), rt, 1 h, 1: 11%,
2: 32%, 3: 36% (4 steps).
primer P2 was 50-32P-radiolabeled and incubated at 37 °C for
60 min in the presence of the various dPTPs and TdT (4 U/reaction).
Reactions with the natural dTTP were also carried out in parallel
for the sake of comparison, since this led to a nicely defined ladder
(with the incorporation of up to 50 nucleotides). The resulting
reaction products were then analyzed by gel electrophoresis (PAGE
20%), as shown in Figure 3.
For all the dPTPs, multiple (i.e. 3–5 nucleosides) incorporations
were observed (lanes 2–4 and 6–8). Moreover, d4PTP 3 revealed to
be the most proficient substrate for the TdT extension reaction. In-
catalysis of the tailing reaction.23 Consequently, when the poly-
merase reaches the modified extended section, the reaction will
stall, which might explain why no more than 5 residues are
appended.
Therefore, the pyrene-deoxynucleoside triphosphates are much
better substrates for the TdT polymerase than for the Klenow frag-
ment. In addition, their tailing efficiencies compare favorably to
that of 20,40-bridged nucleoside triphosphates, where only single
deed, at a concentration of 100 lM, mainly 4 additional nucleo-
tides were incorporated (lane 8) with minor bands corresponding
to 5 and 3 added residues. On the other hand, the reaction at a
higher dPTP concentration (200 lM) led to the incorporation of 5
1 2 3 4 5 6 7 8 9
extra nucleotides (lane 2) with only a minor (ꢀ15%, see Table S1,
Supplementary data) band corresponding to the primer extended
with 4 d4Ps units. In addition, at a lower triphosphate concentra-
tion, d1PTP 1 mainly led to the addition of 4 residues (lane 6), while
an uneven distribution pattern is observed for d2PTP 2 under the
same conditions (lane 7 and Table S1). Moreover, higher triphos-
phate concentrations coerce a more even incorporation distribu-
tion for both d1P (lane 4) d2P (lane 3 and Table S1), with the
main products corresponding to the addition of 4 and 5 extra res-
idues. Finally, the terminal transferase necessitates at least three
deoxynucleotide residues on the primer strand for an efficient
P
Time
Time
Time
Time
Time
B
A
D
E
C
Figure 2. Gel image (PAGE 20%) of the running start primer-extension reactions:
(A) d4PTP 3, dATP, dCTP, dGTP, dTTP; (B) d2PTP 2, dATP, dCTP, dGTP, dTTP; (C) d1PTP
1, dATP, dCTP, dGTP, dTTP; (D) dATP, dCTP, dGTP, dTTP; (E) Control full length
extension product with primer P2, template T2 and only the natural dNTPs; P: 32P-
labeled primer P2; Time points (shown from right to left) were: 1, 30, 60, 120 min.
Figure 3. Gel image (PAGE 20%) of the TdT-mediated polymerization reactions.
Lane 1: dTTP (200
d1PTP (200 M); lane 5: 32P-labeled primer; lane 6: d1PTP (100
(100 M); lane 9: dTTP (100 M).
M); lane 8: d4PTP (100
l
M); lane 2: d4PTP (200
l
M); lane 3: d2PTP (200
l
M); lane 4:
l
l
M); lane 7: d2PTP
l
l
l

4430
M. Hollenstein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4428–4430
insertions were observed.24 Surprisingly, the dPTPs are only
slightly inferior TdT-substrates than some base modified dNTPs,
where only in the case of some dGXTPs, long tails of extra nucleo-
tides were observed.25 Finally, all these dPTPs had better tailing
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In summary, application of the Ludwig-Eckstein method led to
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M.H. was supported by a Grant from the Swiss National Science
Foundation(Grant no PZ00P2_126430). F.W. acknowledges support
from an EU Marie-Curie fellowship. M.H. thanks Dr. A. Calabretta
and Ms. C. Smith for fruitful discussions.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
101.